cells Article Compositional Features of Distinct Microbiota Base on Serum Extracellular Vesicle Metagenomics Analysis in Moderate to Severe Psoriasis Patients Chih-Jung Chang 1,2, Jing Zhang 3, Yu-Ling Tsai 4, Chun-Bing Chen 2,5,6,7,8,9 , Chun-Wei Lu 2,6,8, Yu-Ping Huo 9, Huey-Ming Liou 9, Chao Ji 3,* and Wen-Hung Chung 1,2,5,6,7,8,9,10,* 1 Medical Research Center and Xiamen Chang Gung Allergology Consortium, Xiamen Chang Gung Hospital, Xiamen 361028, China; [email protected] 2 Drug Hypersensitivity Clinical and Research Center, Department of Dermatology, Chang Gung Memorial Hospital, Linkou, Taoyuan 333423, Taiwan; [email protected] (C.-B.C.); [email protected] (C.-W.L.) 3 Department of Dermatology, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China; [email protected] 4 Department of Pathology, Tri-Service General Hospital, Taipei 114202, Taiwan; [email protected] 5 Cancer Vaccine and Immune Cell Therapy Core Laboratory, Department of Medical Research, Chang Gung Memorial Hospital, Linkou, Taoyuan 333423, Taiwan 6 College of Medicine, Chang Gung University, Taoyuan 333323, Taiwan 7 Whole-Genome Research Core Laboratory of Human Diseases, Chang Gung Memorial Hospital, Keelung 20445, Taiwan 8 Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan 333323, Taiwan Citation: Chang, C.-J.; Zhang, J.; Tsai, 9 Department of Dermatology, Xiamen Chang Gung Hospital, Xiamen 361028, China; Y.-L.; Chen, C.-B.; Lu, C.-W.; Huo, [email protected] (Y.-P.H.); [email protected] (H.-M.L.) Y.-P.; Liou, H.-M.; Ji, C.; Chung, W.-H. 10 School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China Compositional Features of Distinct * Correspondence: [email protected] (C.J.); [email protected] or [email protected] (W.-H.C.); Microbiota Base on Serum Tel.: +86-18651619908 (C.J.); +886-3-3281200 (ext. 8494) (W.-H.C.) Extracellular Vesicle Metagenomics Analysis in Moderate to Severe Abstract: The bacterial microbiota in the skin and intestine of patients with psoriasis were different Psoriasis Patients. Cells 2021, 10, 2349. compared with that of healthy individuals. However, the presence of a distinct blood microbiome https://doi.org/10.3390/ in patients with psoriasis is yet to be investigated. In this study, we investigated the differences in cells10092349 bacterial communities in plasma-derived extracellular vesicles (EVs) between patients with moderate to severe psoriasis (PSOs) and healthy controls (HCs). The plasma EVs from the PSO (PASI > 10) Academic Editor: (n = 20) and HC (n = 8) groups were obtained via a series of centrifugations, and patterns were Maria Cristina Curia examined and confirmed using transmission electron microscopy (TEM) and EV-specific markers. The taxonomic composition of the microbiota was determined by using full-length 16S ribosomal Received: 1 July 2021 Accepted: 31 August 2021 RNA gene sequencing. The PSO group had lower bacterial diversity and richness compared with Published: 8 September 2021 HC group. Principal coordinate analysis (PCoA)-based clustering was used to assess diversity and validated dysbiosis for both groups. Differences at the level of amplicon sequence variant (ASV) Publisher’s Note: MDPI stays neutral were observed, suggesting alterations in specific ASVs according to health conditions. The HC group with regard to jurisdictional claims in had higher levels of the phylum Firmicutes and Fusobacteria than in the PSO group. The order published maps and institutional affil- Lactobacillales, family Brucellaceae, genera Streptococcus, and species Kingella oralis and Aquabacterium iations. parvum were highly abundant in the HC group compared with the PSO group. Conversely, the order Bacillales and the genera Staphylococcus and Sphihgomonas, as well as Ralstonia insidiosa, were more abundant in the PSO group. We further predicted the microbiota functional capacities, which revealed significant differences between the PSO and HC groups. In addition to previous studies Copyright: © 2021 by the authors. on microbiome changes in the skin and gut, we demonstrated compositional differences in the Licensee MDPI, Basel, Switzerland. microbe-derived EVs in the plasma of PSO patients. Plasma EVs could be an indicator for assessing This article is an open access article the composition of the microbiome of PSO patients. distributed under the terms and conditions of the Creative Commons Keywords: psoriasis; extracellular vesicle; microbiota; full-length 16S rRNA Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cells 2021, 10, 2349. https://doi.org/10.3390/cells10092349 https://www.mdpi.com/journal/cells Cells 2021, 10, 2349 2 of 16 1. Introduction Chronic plaque psoriasis or psoriasis vulgaris is the most common form of psoriasis and has clinical features characterized by recurrent episodes of red and scaly skin plaques located on the scalp, elbows, knees, umbilicus, and lumbar region [1]. Patients with psoriasis not only suffer from inflammatory skin diseases, but are also often burdened with comorbidities, such as hyperlipidemia, type 2 diabetes, obesity, inflammatory bowel disease (IBD), and cardiovascular disease [2]. Evidently, psoriasis is a systemic inflammatory disease, and it is crucial to develop new potential therapeutic strategies. Extracellular vesicles (EVs) secreted by prokaryotes and eukaryotes are responsible for cell–cell communication [3]. The diverse cargos, size, and concentration of EVs reflect the state of their cells of origin and serve as biomarkers of various pathological condi- tions [4]. The lipid bilayer membrane of EVs from donor cells carries original contents, such as DNA, RNA (microRNA, mRNA, long noncoding RNAs), proteins, and lipids, which are subsequently deposited into the cytosol of recipient cells to trigger modulation of immunity [5]. Recently, EVs from immune cells have been shown to be involved in the pathogenesis of psoriasis. Many studies have shown that Th17 cells and the Th17 pathway play a more and more important role in the occurrence and development of psoriasis. As a downstream effector molecule of Th17 cells, interleukin-17A (IL-17A) can induce fibroblasts and epithelial cells to secrete a variety of cytokines and promote local inflamma- tion [6]. Lin et al. demonstrated that the increased number of mast cells and neutrophils in psoriatic lesions contributes to the release of the pathogenic cytokine IL-17A through EV formation [7]. Claire et al. detected an increase in IL-17A-producing exosomes in psoriasis patients [8]. Drugs targeting free IL-17 and its receptor are nowadays the most effective treatment in managing psoriasis [9,10].The release of EVs is a bidirectional communication between keratinocytes and neutrophils in the inflammatory state of psoriasis [11,12]. Pso- riatic keratinocytes induce the polarization of Th1/Th17 cells via specific microRNAs in secreted EVs and further promote psoriasis development [13]. In addition to immune-cell secreted EVs as pathogenic factors for psoriasis, encapsuled microRNA is considered a biomarker for psoriasis [14,15]. EVs from bacteria, called membrane vesicles (MVs) or outer membrane vesicles (OMVs) that originated from Gram-negative and Gram-positive bacteria, respectively, also contain microorganism-associated molecular patterns (MAMPs), such as nucleic acid, lipopolysaccharides (LPS), peptidoglycan, or toxin to modulate im- munity [16]. Those MAMPs are recognized by different families of pattern recognition receptors (PRRs) expressed by both immune and nonimmune cells in the host and further trigger immunomodulatory signaling [17]. The EVs are characterized as transportation in circulation systems regardless of the EVs secreted from eukaryotes and prokaryotes. Dysbiosis of the skin or gut microbiota is a risk factor that contributes to inadequate innate and adaptive immunity [18,19]. The gut–skin axis is a critical factor in the pathogenesis of psoriasis. Microbe-derived EVs are detectable in the body fluids and play an important role in microbe–host interaction [20]. Pathogen-derived EVs have been demonstrated to regulate disease development, such as in neutrophilic pulmonary inflammation and atopic dermatitis [21]. Beneficial microbe-derived EVs have been found to ameliorate inflamma- tion and affect distal host cells [22,23]. An in-depth analysis of EV metagenome could be an excellent means to systemically detect the bacteria–host relationship [24]. Studies have shown that the gut or skin microbiota play a critical role in psoriasis; however, no study has analyzed systemic microbiome factors. This study investigated the diversity and abundance of microbial EVs in sera collected from PSO patients and healthy controls (HCs). Full-length 16S metagenomic analysis identified biologically and statistically significant bacterial EV taxa as a diagnostic model. 2. Materials and Method 2.1. Sample Collection Serum specimens were collected from PSO patients (PASI > 10) and HCs who had neither skin diseases nor any significant underlying diseases, such as metabolic syndrome, Cells 2021, 10, 2349 3 of 16 intestinal disease, and cancers. None of the participants used probiotics, systemic antibi- otics, or steroids within 14 days prior to the study. All the participants provided written informed consent for the publication of their case details. The study was approved by the Ethics Committee of Xiamen Chang Gung Hospital (approval number XMCGIRB2020039, date of approval: 16 December 2020). 2.2. Isolation of EVs Plasma